The present invention relates generally to methods for optimizing production of recombinant antibodies and nucleic acid sequences which code for novel light chain proteins, the later of which are used in conjunction with the inventive methods. More particularly, the novel light chains which demonstrate the efficacy of the inventive methods can also be utilized in conjunction with a panel for comparing the amino acid sequences of amyloid-associated unmunoglobulin light chains to sequences of non-pathogenic light chains. In such a manner protein regions responsible for self-association and fibril formation can be identified and, ultimately, provided a basis for rational drug design.
Detailed analyses of the structures and biophysical properties of unmunoglobulin molecules have, over the years, probed many aspects of immunoglobulin function, particularly antibody-antigen interactions and effector functions. See Padlan, Anatomy of the Antibody Molecule, Mol. Immunol. 31:169-217, 1994. Immunoglobulin genes have been cloned and altered by mutagenesis to investigate effects of the changes on biological activities, and synthetic immunoglobulin genes have been generated for the production of unique antibody reagents for medical and diagnostic purposes. Another important area of immunoglobulin biology and analysis is the structural characterization of pathological protein deposits formed in humans when plasma cell dyscrasias result in excess production of immunoglobulin protein chains.
Amyloidosis is a severe pathological condition in which deposits of extracellular protein form insoluble fibers in tissues. Amyloid fibers are non-branching fibrils of diameter 70-100 A. Birefringence of bound Congo Red dye demonstrates that proteins within an amyloid fibril are highly ordered. The fibrils are virtually insoluble, except under extremely denaturing conditions, suggesting a large number of molecular interactions contribute to amyloid stability. These tissue deposits impair organ function, and extensive amyloid deposition can lead to death due to organ failure. Many different types of proteins are known to form amyloids, but any particular amyloid deposit contains an essentially homogeneous protein core of primarily xcex2-sheet structure. See Stone, Amyloidosis: A Final Common Pathway for Protein Deposition in Tissues, Blood 75:531-545, 1990. In light chain amyloidosis (AL-amyloidosis) a monoclonal immunoglobulin light chain forms the amyloid deposits. See Glenner et al., Amyloid Fibril Proteins: Proof of Homology with Immunoglobulin Light Chains by Sequence Analyses, Science 172:1150-1151, 1971. Amyloid fibrils from patients suffering AL-amyloidosis occasionally contain only intact light chains, but more often they are formed by proteolytic fragments of the light chains which contain the VL domain and varying amounts of the constant domain, or by a mixture of fragments and fuil-length light chains. Not all light chains from plasma cell dyscrasias form protein deposits; some circulate throughout the body at high concentrations and are excreted with the patients urine without pathological deposition of the protein in vivo. See Solomon, Clinical Implications of Monoclonal Light Chains, Semin. Oncol. 13:341-349, 1986; Buxbaum, Mechanisms of Disease: Monoclonal Immunoglobulin Deposition, Amyloidosis, Light Chain Deposition Disease, and Light and leavy Chain Deposition Disease, Hematol/Oncol. Clinics of North America 6:323-346, 1992; and Eulitz, Amyloid Formation from Immunoglobulin Chains, Biol. ChenL Hoppe-Seyler 373:629-633, 1992.
In some types of hereditary amyloidoses, single amino acid changes in normal human proteins are responsible for amyloid fibril fornation See Natvig et al., Amvloid and Amyloidosis 1990. Dordrecht, The Netherlands: Kluwer Academic Publishers, 1991, and references cited therein. It is unlikely, however, that any single amino acid position or substitution will fully explain the many different immunoglobulin light chain sequences associated with AL-amyloidosis. Rather, several different regions of the light chain molecule may sustain one or more substitutions which affect a number of biophysical characteristics, such as dimer formation, exposure of hydrophobic residues, solubility, and stability.
Increased dimerization, for example, may promote amyloid deposition of a protein. It has been shown that an extremely high proportion of rREC occurs as dimers, even at very low concentrations of the recombinant protein. The calculated dimerization constant for rREC is xcx9c107, approximately two orders of magnitude higher than that of rLEN. The dimerization constant of rLEN, xcx9c5xc3x97105 Mxe2x88x921, is in the range of self-association constants observed for other human immunoglobulin light chains. For KI protein AU, for example, a value of 6.6xc3x97104 Mxe2x88x921 was experimentally determined (see Maeda et al., Kinetics of Dimerization of the Bence-Jones Protein AU, Biophys. Chem. 9:57-64, 1978); values from xcx9c103 Mxe2x88x921 to xcx9c106 Mxe2x88x921 were estimated for a large number of human immunoglobulin KI light chains (see Stevens et al., Self-association of Human Immunoglobulin xcexaI Light Chains: Role of the Third Hypervariable Region, Proc. Natl. Acad. Sci. USA 77:1144-1148, 1980); and values of xcx9c2.5xc3x97105 Mxe2x88x921 to xcx9c5.0xc3x97106 Mxe2x88x921 were calculated for variant REI VKI domains. Computer simulation of rREC dimerization, however, yield a dimerization constant of 5xc3x97107 Mxe2x88x921.
It has been suggested that unusual amino acids within the inner xcex2-sheets which form the contact regions at the dimer interface may be responsible for increasing dimer stability of amyloidogenic light chains, thereby promoting fibril formation. See Dwulet et al., Amino Acid Sequence of a xe2x8ax96 VI Primary (AL) Amyloid Protein. Scand. J. Immunol. 22:653-660, 1985; Liepnicks et al., Comparison of the Amino Acid Sequences of ten kappa I Amyloid Proteins for Amyloidogenic Sequences, In: Natvig J B, et al. Amyloid and Amyloidosis 1990. Dordrecht, The Netherlands: Kluwer Academic Publishers, pp. 153xe2x80x94156, 1991; and Aucouturier et al., Complementary DNA Sequence of Human Amyloidogenic Immunoglobulin Light-Chain Precursors. Biochem. J. 285:149-152, 1992. The positional effect of amino acids is illustrated by two unanticipated features in the crystallographic structures of naturally occuhing light chains obtained from human patients. In one structural investigation study, a glutamine residue at position 38 was observed to have been replaced by a histidine residue in the Bence-Jones protein Loc. The crystal structure of the protein crysted from ammonium sulfate differed from that of the protein crystallized from distilled water. The quaternary interactions exhibited by the protein in the two crystal forms were sufficiently different to suggest fimdalmentally different interpretations of the structural basis for the function of this protein. See Schiffer et al, The Structure of a Second Crystal Form of Bence Jones Protein Loc: Strikingly Different Domain Associations in Two Crystal Forms of a Single Protein, Biochemistry 28:4066-4072, 1989. In a second crystallographic analysis, a highly conserved tyrosine residue at position 36 was observed to have been replaced by a phenylalanine residue, the structural differences again suggesting an altered quaternary interaction. See Huang et al., Novel Immunoglobulin Variable Domain Interaction is Observed, American Crystallographic Association Meeting, 1993, p 127. Notwithstanding findings of this sort, the stability of amyloidogenic dimers is not fully understood: The sequence of the amyloid protein REC differs from that of LEN primarily at CDR residues and not at residues comprising the xcex2-sheet framework.
Nonetheless, there has been great interest in deteimining the sequences of amyloid-associated immunoglobulin light chains and comparing them to sequences of non-pathogenic light chains to identify regions of the proteins responsible for self-association and fibril formation. A substantial number of sequences of amyloidogenic immunoglobin in light chains have been obtained either by direct amino acid sequencing of protein isolated from patient urine or from amyloid deposits or by nucleotide sequencing of cDNAs cloned from plasma cells of patients with AL-type amyloidosis, but no particular common sequences have been identified as obviously correlating with the pathogenic properties of the amyloid-associated light chains. See Natvig et al., supra; Aucouturier et al., supra, and references cited therein.
Another approach to understanding the molecular differences between non-pathogenic and amyloidogenic light chais is to probe the in vivo disease process of protein deposition by in vitro exammation of various biochemical and biophysical properties of light chain proteins which either are xe2x80x9cbenignxe2x80x9d or form protein deposits in vivo. See Solomon, Bence Jones Proteins: Malignant or Benign? N. Engl. J. Med. 306:605-607, 1982; and Myatt et al., Pathogenic Potential of Human Monoclonal Immunoglobulin Light Chains: Relationship of in vitro Aggregation to in vivo Organ Deposition, Proc. And. Acad. Sci. USA 91:3034-3038, 1994. Characterization of the chemical anctphysical properties of amyloid-associated immunoglobulii light chains has been difficult, however. Because these light chains accumulate in insoluble extracellular deposits, it is generally difficult to obtain the relevant light chain protein from patient serum or urine in quantities sufficient for analyses. Solubilintion of light chain proteins from amyloidladen tissue obtained post mortem requires harsh chemical treatments and provides only a limited, non-replenishable protein supply.
A somewhat effective approach has been to apply recombinant bacterial techniques enroute to both benign immunoglobulin light-chain domains and those known to produce pathological deposits. In such a manner, large quantities of light chain proteins are available, such that their biophysical and biochemical properties can be thoroughly studied. Comparisons of the benign and pathological light chains can provide the basis for production of mutated proteins modified at particular residues for in vitro analysis of the effects of these mutations on various biophysical characteristics.
Traditionally, antibodies have been obtained by the immunization of animals, such as goats and rabbits, and subsequent purification from the animal blood. The quality of the antisera intermittently obtained from a single animal was variable, and the characteristics of the antisera obtained from any two animals were often different. Methods were subsequently developed which allowed the fusion of an antibody-producing lymphocyte with an immortal myeloma cell; i.e., a cancerous lymphocyte capable of continuous replication. Such hybridomas became sources of chemically homogeneous monoclonal antibodies which allowed for more predictable and controllable technological application More recently, techniques were developed for the transfer of antibody genes into bacteria. These recombinant bacteria produce antibodies identical to those produced by the aniimal from which the gene was obtained.
However, recombinant techniques are not without problems and deficiencies. Effective commercial use of recombinant antibodies for immunodiagnostic, immunotherapeutic, or other applications in industrial, environmental and/or agricultural fields requires maiial yields. In many cases, even where synthesized by bacteria, the productivity of functional antibody is erratic and is frequently too low to be useful. Less than optimal productivity is often related to diminshed functional Fab and Fv assemblies, resulting from homologous dimer self-association.
It is, therefore, an object of this invention to provide a method for increased yields of synthesized recombinant antibody, overcoming the problems and deficiencies of the prior art, including those discussed above.
It is also an object of this invention to provide a method for increased yields of synthesized recombinant antibody, utilizing, inter alia, improved control of variables related to antibody assembly.
Another object of this invention is to provide novel light chains which demonstrate the efficacy of the inventive methods.
Another object of this invention is to characterize the molecular interactions involved in amyloid fibril formation, stability, and insolubility enroute to the development of effective therapies.
Another object of this invention is to provide novel light chains or fragments thereof for use in conjunction with a panel for comparing the amino acid sequences of amyloid-associated immunoglobulin light chain sequences of non-pathogenic light chains.
Another object of this invention is to enhance assembly of functional variable domain fragments.
Another object of this iiveiion is to provide a method for light chain and heavy chain variable domain complex formation, such that the complex is capable of binding antigen.
Another object of this invention is to provide a method for antigen binding fragment fotmation.
Another object of this invention is to favorably influence the rate of variable domain fcragent assembly, increasing the concentration of heavy and/or light chain variable domain, suce that the fragment yield is increased.
Another object of this invention is to lower the equilibrium constant for one or both homologous variable domain associations and/or reduce the incidence of such reactions in competition with heterologous dimerizations.
Another object of this invention is to cbncombinantly increase heterologous associations and decrease homologous associations.
Another object of this invention is to increase the yield of recombinant Fv and Fab assemblies through modification of the amino acid sequence in the interfacial segments of the light and heavy chain variable domains.
Another object of this invention is to increase the yield of antigen binding fragment and variable domain fragnent assemblies by altering the free energy requirements of dimerizaton and promoting productive variable domain associations.
Another object of this invention is to alter the amino acid sequence of light and heavy chain variable domains to provide energetically favorable contacts across the variable domain interface.
Another object of this invention is to increase productive associations through modification of variable domain affinity and/or geometry by rational substitution of interfacial amino acids.
Another object of this invention is to apply the principles and/or precepts underline any and each of the foregoing objects to modification of nucleic acid sequences coding for antibody subunits of light and heavy polypeptides and the expression of the modified sequences.
Other objects, features and advantages of the present invention will be readily apparent from the following description of the preferred embodiments thereof, taken in conjunction with the accompanying examples, figures, and sequences.